1. Field of the Invention
The present invention relates generally to methods and apparatus for detecting analytes in liquid samples. More particularly, the present invention relates to assays for detecting enzymes and components of enzyme pathways where both the time and temperature of the assay are controlled.
Enzymes and enzymatic pathways play an important role in medicine and are the subject of many clinical tests. Examples of tests for single enzymes include tests for amylase, creatine kinase, alanine aminotransferase, aspartate aminotransferase, streptokinase, and thrombin. Examples of tests for enzymatic pathways include the prothrombin time test, and the activated partial thromboplastin time test. These later tests measure the enzymatic pathways involved in the extrinsic and intrinsic blood coagulation systems.
Tests involving enzymatic reactions tend to be technically demanding. Enzymes and enzymatic pathways are typically assayed by measuring the rate at which the enzyme or enzymatic pathway in question converts a particular enzymatic substrate into its product. Such rate measurements require precise test timing since timing errors are directly translated into errors in the calculated amount of enzyme or component in an enzyme pathway.
Temperature control is also critical since most enzymes have reaction rates that change dramatically as a function of temperature. Typically, higher temperatures produce a higher reaction rate, and lower temperatures produce a lower reaction rate. Enzymatic pathways, consisting of a number of temperature sensitive enzymatic steps, are often extremely temperature sensitive as a result of the cascade effect.
Because of these technical demands, most enzyme and enzymatic pathway tests have tended to be complex, with performance generally limited to clinical laboratories. While such centralized testing may be adequate for infrequent, or routine medical conditions, visiting a doctor's office or a clinic on a regular basis for frequent or emergency medical tests is less acceptable. Thus there exists a need for convenient and simple tests, that can be performed by unskilled users for the measurement of enzymes and enzyme pathways.
A variety of simplified "test-strip" assays have been developed to allow semi-skilled and unskilled users to detect analytes, such as pregnancy hormones, cholesterol, and glucose in urine, blood, and other patient samples. These test strip assays are most useful with non-enzymatic analytes where detection does not vary with minor fluctuations in test time or temperature. As previously discussed, enzymatic reactions are less tolerant, and require more precise control over these variables, generally rendering them unsuitable for use in the home or other non-clinical environment.
One such test strip for performing blood glucose analysis, available from LifeScan Inc., Milpitas, Calif., relies on applying a drop of blood to a polyamide membrane having impregnated reagents which produce a chromogenic reaction in response to the glucose level in applied blood. Simplified low-cost tests such as this are often referred to as "home tests" to designate the fact that they have achieved a price and simplicity level that would allow widespread adoption in non-professional settings.
For these reasons, it would be desirable to provide simplified assays, test articles, and test systems for detecting problematic analytes, such as enzymes and components of enzyme pathways in a variety of patient samples, such as blood, urine, and the like. In particular, the test articles and test systems should permit simplified assay protocols, preferably allowing for an automatic timing cycle which is initiated as soon as a sample is applied to a test article. The test articles and test systems should optionally also facilitate providing precise temperature control of a test region on the article, preferably without the need to enclose the test article in a heated chamber or other structure which limits the user access. The assays, test articles, and test systems should be readily usable with small sample volumes, particularly with small blood volumes such as a single blood drop. The test articles should further inhibit loss of the patient sample from the test article by evaporation or other means, particularly when using very small sample volumes. The test article and test system should still further provide for monitoring of the presence of sample within the test article and be able to warn the user when excessive amounts of sample have been lost. Such test articles and test systems should be both easy to manufacture and easy to use, preferably being producible at relatively low costs.
2. Description of the Background Art
Assay devices which detect the presence of an analyte based on the enzymatic conversion of a substrate to a visible or detectable product within an absorptive matrix are described in U.S. Pat. Nos. 5,059,525; 5,059,394; 4,256,693; 4,935,346; 3,791,933; and 3,663,374. Analytical apparatus having means for detecting sample application are described in U.S. Pat. Nos. 5,049,487 and 4,420,566. The '487 patent describes a timing circuit which is triggered by detecting a change in reflectance caused by wetting of a porous matrix. The '566 patent describes the measurement of light absorbance to confirm that a liquid sample has been applied to a slide prior to analysis. Systems for controlling the temperature of analytical test substrates are described in U.S. Pat. Nos. 4,720,372; 4,219,529; and 4,038,030. Analytical test substrates comprising ion selective electrodes are described in U.S. Pat. Nos. 4,171,246 and 4,053,381.